Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that leads to severe disability and premature death in young men. As DMD is caused by the absence of dystrophin, therapeutic development has focused on strate...Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that leads to severe disability and premature death in young men. As DMD is caused by the absence of dystrophin, therapeutic development has focused on strategies to restore dystrophin expression. These include readthrough of premature stop codons, exon skipping to restore the reading frame, and gene therapy. The first two methods are mutation-specific, benefiting only subsets of patients, whereas gene therapy could treat all individuals with DMD. Immunogenicity of dystrophin may challenge these efforts. The immune system can recognize dystrophin as a neo-antigen, just as it can recognize newly arising antigens present on mutated cells. An in-depth evaluation of anti-dystrophin immune response as a factor affecting the treatment effectiveness is needed. Key questions include the underlying mechanisms of immunity induction by antigenic epitopes of the re-expressed dystrophin, the impact of such responses on the therapeutic efficacy, and the role of patient-specific risk factors, such as preimmunization due to revertant fibres, chronic muscle inflammation, pre-existing T lymphocytes reactive to dystrophin, which avoided deletion in dystrophic thymus, or antigen cross-reactivity. Patients' immune status assessment before treatment may help mitigating anti-dystrophin responses. Exploring potential therapeutic strategies to enhance treatment outcomes is also essential: Since DMD can be diagnosed at birth, early dystrophin re-expression could prevent damage and also potentially induce neonatal tolerance. In older patients, carefully managed immunosuppression and tolerogenic protocols could pave the way for more successful dystrophin replacement therapies.
The present study examines the safety profile of intravenous onasemnogene abeparvovec gene therapy in a real-world setting, both alone or in combination with intrathecal antisense oligonucleotide nusinersen therapy in tw...The present study examines the safety profile of intravenous onasemnogene abeparvovec gene therapy in a real-world setting, both alone or in combination with intrathecal antisense oligonucleotide nusinersen therapy in two cohorts of patients with spinal muscular atrophy (SMA). The first cohort included eight presymptomatic infants treated solely with onasemnogene abeparvovec, while the second cohort comprised six symptomatic infants receiving onasemnogene abeparvovec and nusinersen co-therapy. All patients received the corticosteroid prednisolone coincident with gene therapy. Circulating alanine aminotransferase (ALT) and aspartate transaminase (AST) levels were measured to determine potential hepatoxicity, the primary focus of this study. Elevated ALT and AST levels were observed in one pre-symptomatic and three symptomatic patients post-treatment. However, all values returned to normal levels within 3 months of onasemnogene abeparvovec injection. Nusinersen treatment received previously or coincident with gene therapy did not impact the transient elevation of liver transaminases. This study highlights the importance of early intervention with molecular treatments for SMA and indicates that prior or coincident treatment with nusinersen is unlikely to impact safety of onasemnogene apoparvovec and could theoretically improve clinical outcomes in symptomatic infants or in those with gene therapy delayed beyond the immediate neonatal period.
Advancements in our understanding of genetic disease and adeno-associated virus has prompted great excitement into the field of AAV-mediated gene therapy, particularly for genetic diseases of the central nervous system,...Advancements in our understanding of genetic disease and adeno-associated virus has prompted great excitement into the field of AAV-mediated gene therapy, particularly for genetic diseases of the central nervous system, including retinal disorders. Despite significant progress, exemplified by the approval of therapies such as Luxturna® and Zolgensma®, a substantial number of therapies remain in pre-clinical or early clinical stages, with many failing to advance to later phases. Whilst the use of animal models to test safety and delivery route efficacy of AAV treatments is imperative, differences in tissue structure and physiology between humans and animal models has restricted precise disease modelling and gene therapy development for many CNS disorders. Alongside the FDA push for non-animal alternative models, researchers are increasingly turning to human-based models, including stem cell-derived organoids, which can offer a more accurate representation of human cellular microenvironments and niches. As such, this review explores the advantages and limitations of brain and retinal organoids as pre-clinical models of disease, with a primary focus on their utility in identifying novel AAV capsids, cell-specific promoters, and their role in recent pre-clinical AAV gene therapy studies.
Adeno-associated viral (AAV) vectors are an ideal platform for gene therapy due to their ability to deliver therapeutic cargos safely and effectively across various target organs. Their low immunogenicity contributes to...Adeno-associated viral (AAV) vectors are an ideal platform for gene therapy due to their ability to deliver therapeutic cargos safely and effectively across various target organs. Their low immunogenicity contributes to long-lasting therapeutic effects. However, recent insights highlight the significance of CpG content within AAV vectors, where unmethylated CpG dinucleotides can trigger a TLR9-mediated immune response, leading to the rapid elimination of transduced cells. Clinical evidence indicates an inverse relationship between CpG content and therapeutic success, with lower CpG counts correlating with sustained transgene expression. Here, we sought to optimize a novel, CpG-rich AAV8 vector, referred to as pVR59, designed for treating lipoprotein lipase deficiency (LPLD). We strategically reduced CpG levels in pVR59, resulting in the development of pNC182, a CpG-depleted vector that maintains therapeutic efficacy. A single intramuscular injection of pNC182 demonstrated comparable effectiveness to pVR59 in normalizing lipemia and hypertriglyceridemia in LPLD mouse models, with a 38% reduction in total CpG count. These findings support the clinical application of pNC182 as a safe, long-lasting AAV gene therapy for LPLD and provide a framework for future AAV vector designs aimed at maximizing therapeutic efficacy while minimizing immunogenic responses in human settings.
Age-related Macular degeneration (AMD) is a major cause of vision loss and is linked to several predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing offers the potential to target negativel...Age-related Macular degeneration (AMD) is a major cause of vision loss and is linked to several predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing offers the potential to target negatively associated SNPs in an allele-specific manner, necessitating the need for a relevant cell model. The ARPE-19 cell line, with its stable monolayer growth and retinal pigment epithelium (RPE) characteristics, serves as an ideal model for AMD studies. Chronic inflammation and complement system dysregulation are implicated in AMD pathogenesis. Most genetic variations associated with AMD are in complement genes, suggesting their regulatory role. In this study, we conducted targeted PCRs to identify AMD-related SNPs in ARPE-19 cells and used CRISPR constructs to assess allele-specific activity. Guide RNA sequences were cloned into an EF-1-driven SpCas9 vector and packaged into lentivirus. Targeting efficiencies were evaluated with TIDE analysis, and allele-specificity was measured with NGS analysis 30 days post-transduction. Our results showed varying targeting efficiencies depending on guide RNA efficacy. For example, TIDE analysis of CFH SNPs rs1061170 and rs1410996 revealed efficiencies of 35.5% and 33.8%, respectively. CFB SNP rs4541862 showed efficiencies from 3% to 36.7%, and rs641153 ranged from 3.4% to 23.8%. Additionally, allele-specific targeting of AMD-related SNPs rs1061170, rs1410996, rs4541862, and rs641153 ranged from 48% to 52% in heterozygous differentiated ARPE-19 cells. These findings demonstrate the potential to manipulate the complement system in an AMD model by targeting disease-associated SNPs in an allele-specific manner, offering a promising therapeutic approach.
Inherited Phosphomannomutase 2 (PMM2) deficiency, also known as PMM2-CDG, is the most prevalent N-linked congenital disorder of glycosylation (CDG), occurring in approximately 1 in 20,000 individuals in certain populatio...Inherited Phosphomannomutase 2 (PMM2) deficiency, also known as PMM2-CDG, is the most prevalent N-linked congenital disorder of glycosylation (CDG), occurring in approximately 1 in 20,000 individuals in certain populations. Patients exhibit a spectrum of symptoms, with neurological involvement being a prominent feature, often manifesting as the initial clinical sign, and can range from isolated neurological deficits to severe multi-organ dysfunction. Given the absence of curative treatments and a high mortality rate before the age of two, alongside considerable lifelong morbidity, there is an urgent need for innovative therapeutic approaches. To address this unmet need, we developed a tamoxifen-inducible Pmm2 knockout (KO) mouse model with widespread tissue deficiency of Pmm2 expression. Characterization of the mouse model to-date revealed distinct neurological phenotypes relevant to PMM2-CDG, as assessed by the Composite Phenotype Scoring System and Open Field Test. Notably, PMM2 augmentation through AAV9-PMM2 gene replacement therapy prevented and halted the disease-relevant neurological phenotypes induced by Pmm2 KO in the animals. These findings underscored the promise of AAV9-PMM2 gene replacement in managing PMM2-CDG.
The success of mRNA vaccines in controlling the COVID 19 pandemic has confirmed the efficacy of synthetically synthesized mRNA in humans and has also provided a blueprint on how to design them in terms of molecular struc...The success of mRNA vaccines in controlling the COVID 19 pandemic has confirmed the efficacy of synthetically synthesized mRNA in humans and has also provided a blueprint on how to design them in terms of molecular structure and cost. We describe a mRNA vector that, unlike linear mRNAs used in current vaccines/therapeutics, does not require a 5' cap to function. The described mRNA vector initiates translation from an internal ribosomal entry site (IRES) and contains specially designed self-folding secondary structures (hairpins) to protect the 5' end against degradation, dramatically improving its stability. The produced mRNA did not require any additional modifications for functionality. The 5' hairpins completely inhibited cap-dependent translation, and all vectors containing them required an IRES to express protein. When this capless mRNA vector was constructed to express the full-length Influenza A membrane protein hemagglutinin (HA), complexed with pre-formed lipid-based nanoparticles, and then injected into mice as a vaccine, it generated high titers of anti-HA antibodies and protected mice against a lethal dose of Influenza A.
Multiple sclerosis (MS) is an immune-mediated chronic inflammatory and neurodegenerative disease of the central nervous system (CNS) affecting more than 2.5 million patients worldwide. Chronic demyelination in the CNS ha...Multiple sclerosis (MS) is an immune-mediated chronic inflammatory and neurodegenerative disease of the central nervous system (CNS) affecting more than 2.5 million patients worldwide. Chronic demyelination in the CNS has an important role in perpetuating axonal loss and increases difficulty in promoting remyelination. Therefore, regenerative, and neuroprotective strategies are essential to overcome this impediment to rescue axonal integrity and function. Neurotrophin 3 (NT-3) has immunomodulatory and anti-inflammatory properties, in addition to its well-recognized function in nervous system development, myelination, neuroprotection, and regeneration. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of experimental autoimmune encephalomyelitis (EAE) mice, the chronic relapsing mouse model of MS, at 3 weeks post EAE induction. Measurable NT-3 levels were found in serum at 7-weeks post gene delivery. The treated cohort showed improved clinical scores and performed significantly better in rotarod, and grip strength tests compared to their untreated counterparts. Histopathologic studies showed improved remyelination and axon protection. These data correlated with reduced expression of the pro-inflammatory cytokines in brain and spinal cord, and increased percentage of regulatory T cells in the spleens and lymph nodes. Collectively, these findings demonstrate the translational potential of AAV-delivered NT-3 for chronic progressive MS.
Genetic switches that allow for precise control over transgene expression timing or levels may improve the safety and expand the use of adeno-associated viral (AAV) vector-based gene therapy technologies. We previously e...Genetic switches that allow for precise control over transgene expression timing or levels may improve the safety and expand the use of adeno-associated viral (AAV) vector-based gene therapy technologies. We previously engineered an efficient RNA switch system that comprises a novel self-cleaving ribozyme (T3H38) and an octaguanidine dendrimer-conjugated morpholino oligonucleotide (v-M8) complementary to the ribozyme. This switch system can be used to efficiently regulate AAV-delivered transgenes with an up to 200-fold regulatory range in mice. However, this switch system has a relatively short induction half-life and only works well when v-M8 was locally but not systemically administered, representing two key limitations of the system. To address these issues, here, we tested replacing the octa-guanidine dendrimer in the v-M8 morpholino oligo with a cell-penetrating peptide (CPP). Two CPP-conjugated morpholino oligos (B-M8 and B-MSP-M8) were synthesized and compared with v-M8 for the induction of T3H38-regulated AAV-luciferase in mice. One of the CPP-conjugated oligos (B-MSP-M8) not only showed significantly improved induction half-life over that of v-M8, but also enabled efficient induction of AAV transgene expression when the oligo was systemically administered. This study improves in vivo performance and broadens the utility of the T3H38 ribozyme-based RNA switch system in gene therapy applications.
Gumusgoz E, Kasiri S, Youssef I
… +11 more, Verma M, Chopra R, Villarreal Acha D, Wu J, Marriam U, Alao E, Chen X, Guisso DR, Gray SJ, Shah BR, Minassian BA
Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy an...Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.
The liver is a unique organ where immunity can be biased toward ineffective response notably in the context of viral infections. Chronic viral hepatitis depends on the inability of the T-cell immune response to eradicate...The liver is a unique organ where immunity can be biased toward ineffective response notably in the context of viral infections. Chronic viral hepatitis depends on the inability of the T-cell immune response to eradicate antigen. In the case of recombinant Adeno-Associated-Virus, used for therapeutic gene transfer, conflicting reports describe tolerance induction to different transgene products while other studies have shown conventional cytotoxic CD8 T cell responses with a rapid loss of transgene expression. We performed a 1 year follow up of 6 non-human primates after all animals received an rAAV8 vector carrying the GFP transgene at doses of 7×10 vg/kg. We report that despite anti-GFP peripheral cellular response and loss of hepatic transgene expression, we were still able to detect persisting viral genomes in the liver until 1-year post-injection. These viral genomes were associated with liver inflammation, fibrosis and signs of CD8 T cell exhaustion, including high expression of PD-1. Our study shows that AAV8-mediated gene transfer can results to loss of transgene expression in liver and chronic inflammation several months after gene transfer.
Osteoarthritis (OA), a prevalent joint disorder, can lead to disability, with no effective treatment available. Interleukin-1 (IL-1) plays a crucial role in the progression of OA, and its receptor antagonist (IL-1Ra), a...Osteoarthritis (OA), a prevalent joint disorder, can lead to disability, with no effective treatment available. Interleukin-1 (IL-1) plays a crucial role in the progression of OA, and its receptor antagonist (IL-1Ra), a natural IL-1 inhibitor, represents a promising therapeutic target by obstructing the IL-1 signaling pathway. This study delivered IL-1Ra via adeno-associated virus (AAV), a gene therapy vector enabling long-term protein expression, to treat knee osteoarthritis (KOA) in animal models. scAAV-oIL-1Ra-I1/2 injected directly into the joint in both MMT/ACLT-induced KOA model rat improved abnormal gait (increasing footprint area and pressure), subchondral bone lesions, and significantly reduced cartilage wear and pathological scores. In the MMT-induced KOA rabbit model, weight-bearing asymmetry (indicating pain) improved after 8 weeks of scAAV-oIL-1Ra-I1/2 administration, and X-ray showed decreased K-L scores (severity grade), reduced cartilage loss, and lower pathology scores compared to untreated animals. Additionally, sex-determining region Y-type high mobility group box 9 (SOX9) was co-delivered with IL-1Ra via AAV in ACLT + MMT-induced KOA rats. The combined treatment significantly alleviated subchondral bone lesions, cartilage destruction, synovial inflammation, and pathological scores, demonstrating superior efficacy compared to either treatment administered alone. Co-delivering IL-1Ra and SOX9 inhibited IL-1 mediated inflammatory signaling, maintained cartilage homeostasis, and promoted its repair in KOA models, suggesting potential for clinical use.
The approved intravenous adeno-associated virus (AAV) therapies are limited by the widespread prevalence of pre-existing anti-AAV antibodies in the general population, which are known to restrict patients' ability to rec...The approved intravenous adeno-associated virus (AAV) therapies are limited by the widespread prevalence of pre-existing anti-AAV antibodies in the general population, which are known to restrict patients' ability to receive gene therapy and limit transfection efficacy in vivo. To address this challenge, we have developed a novel recombinant human immunoglobulin G degrading enzyme KJ103, characterized by low immunogenicity and clinical value for the elimination of anti-AAV antibodies in gene transfer. Herein, we conducted two randomized, blinded, placebo-controlled, single ascending dose Phase I studies in China and New Zealand, to evaluate the pharmacokinetics, pharmacodynamics, safety and immunogenicity of KJ103 in healthy volunteers. The results confirmed that KJ103 rapidly reduced IgG and maintained plasma IgG at low levels for one week. Dose of KJ103 ranging from 0.01 to 0.40 mg/kg had a favorable safety and tolerability profile across diverse ethnic and gender groups. KJ103 demonstrated a lower incidence of pre-existing anti-drug antibodies (ADAs) compared to currently approved human IgG degrading enzyme Imlifidase, with most induced ADAs predominantly reverting to baseline six months after administration. These properties are ideal for the management of immune disorders, rejection responses, and immunotherapies where pre-existing antibodies can reduce efficacy. Furthermore, we tested AAV2 neutralizing antibodies to confirm the potential utility of KJ103 in enhancing gene therapy.
Spinal muscular atrophy (SMA) is a progressive disease that affects motor neurons, with symptoms usually starting in infancy or early childhood. Recent breakthroughs in treatments targeting SMA have improved both lifespa...Spinal muscular atrophy (SMA) is a progressive disease that affects motor neurons, with symptoms usually starting in infancy or early childhood. Recent breakthroughs in treatments targeting SMA have improved both lifespan and quality of life for infants and children with the disease. Given the impact of these treatments, it is essential to develop methods for managing treatment-induced changes in disease characteristics. Zolgensma® is the first effective and approved gene therapy for SMA caused by biallelic mutation in the SMN1 gene. In three children with SMA treated with Zolgensma®, neuronal, glial, inflammation, and vascular markers in the plasma exhibited a quicker response, emphasizing their potential as valuable biomarkers of treatment efficacy in clinical trials. We chose the novel Nucleic acid Linked Immuno-Sandwich Assay, to investigate a predefined panel of neuroinflammatory markers in plasma samples collected from SMA patients at baseline and six months after Zolgensma® treatment. We identified a set of novel targets whose levels differed between pre and post Zolgensma® treatment group and that were responsive to treatment. Even though our results warrant validation in larger SMA cohorts and longer follow-up time, they may pave the way for a panel of responsive proteins solidifying biomarker endpoints in SMA clinical trials.
Targeted gene delivery to vascular smooth muscle cells (VSMCs) could prevent or improve a variety of diseases affecting the vasculature and particularly the aorta. Thus, we aimed to develop a delivery vector that efficie...Targeted gene delivery to vascular smooth muscle cells (VSMCs) could prevent or improve a variety of diseases affecting the vasculature and particularly the aorta. Thus, we aimed to develop a delivery vector that efficiently targets VSMCs. We selected engineered adeno-associated virus (AAV) capsids from a random AAV capsid library and tested the top enriched motifs in parallel screening through individual barcoding. This approach allowed us to distinguish capsids that only transduce cells based on genomic DNA (gDNA) from those also mediating transgene expression based on transcribed cDNA reads. After three rounds of selection on primary murine VSMCs (mVSMCs), we identified a novel targeting motif (RFTEKPA) that significantly improved transduction and gene expression efficiency over AAV9-wild type (WT) and increased expression in mVSMCs by 70% compared to the previously identified SLRSPPS peptide. Further analysis showed that the novel motif also improved expression in human aortic smooth muscle cells (HAoSMCs) and human aortic tissue ex vivo up to threefold compared to SLRSPPS and approximately 70-fold to AAV9-WT. This high cross-species transduction efficiency makes the novel capsid motif a potential candidate for future clinical application in vascular diseases.